Antimicrobial Potential of Extract from a Pseudomonas aeruginosa Isolate

Microorganisms are one of the main sources of antimicrobial agents and over 50% of antibiotics currently used in hospitals are metabolites from microbes. This study aimed to isolate microorganisms from the Dompoase landfill site, Kwame Nkrumah University Physics Garden, Kosiko River, and Ada Foah seashore of Ghana and screen their metabolites for antimicrobial activity. Forty-eight (48) microorganisms were isolated and their metabolites were screened against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Vibrio cholerae, Salmonella typhi, Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus mirabilis, and Candida albicans using the agar well diffusion method. Ten (10) of the isolates exhibited antimicrobial activity. Isolate DO5, identified as P. aeruginosa isolate, from the Dompoase landfill site was selected for fermentation because it exhibited the highest activity against all the test organisms. DO5 produced optimum antimicrobial activity when fermented for 11 days at 30°C. In the agar diffusion method, the extract of isolate DO5 recorded zones of inhibition ranging between 11.67 ± 0.23 and 21.50 ± 0.71 mm. The MIC and MBC recorded for the DO5 extract ranged from 3.13–25.0 mg/mL and from 6.25–50.0 mg/mL, respectively. Column chromatography analysis yielded eight (8) subfractions from the DO5 extract. IR analysis revealed the presence of functional groups such as alcohols, esters, and hydrocarbons in the fractions. GC-MS analysis identified nine compounds that have been reported to have antimicrobial agents. The DO5 metabolites stand the chance to be developed into potent antibiotics for infection treatment.


Introduction
Antibiotic resistance is one of the most pressing global health issues today. Antibiotic resistance mechanisms frequently occur as a result of misuse of antibiotics in medicine and agriculture in both industrialized and developing nations, posing a threat to modern medicine by reducing the efficacy of therapeutically relevant antibiotics [1][2][3]. e overuse of antibiotics has put bacteria under long-term selective pressure, resulting in antibiotic resistance, which means the bacteria will be tough to kill. Antibiotic resistance genes (ARGs) are the key functional elements in antibioticresistant bacteria and they can move between microbes via horizontal gene transfer [4,5].
Antibiotic and antimicrobial resistance (AMR) has resulted in increased morbidity and death as a result of treatment failures, as well as higher health-care expenses [1,2]. Research has it that AMR is expected to become the major cause of death in the world by 2050, with 10 million fatalities every year. As indicated in the 2014 O'Neill report, which was commissioned by the UK government, AMR is anticipated to have a detrimental impact on the global economy, forecasting a decline in global GDP of 2.5% to 3% (up to $100 trillion) between 2014 and 2050 [1]. us, antibiotic resistance has been dubbed "the silent tsunami facing modern medicine" [4].
Without the invention of novel antibiotics, and as resistance develops, we may find ourselves in a situation similar to that which existed prior to the advent of antibiotics, where routine medical procedures become extremely risky as a consequence of failure to prevent or treat infections that are presently simple to handle [1]. e broad-spectrum beta-lactamase (ESBL) enzymes found in Enterobacteriaceae have also aggravated the global antibiotic-resistance menace. ese ESBL enzymes hydrolyze and inactivate beta-lactam antibiotics such as cephalosporins and also enhance microbial resistance to quinolones, aminoglycosides, and sulfonamides leading to the emergence of multidrug resistance (MDR) [5].
is menace has therefore necessitated the search for more potent metabolites from microorganisms that can be developed into antibiotics as treatment options for resistant bacteria infections [5]. Streptomyces, Bacillus, Cephalosporium, and Penicillium which are known producers of antibiotics are being studied for antibiotics production globally [6,7]. Actinomycetes have also been reported to be producers of many biologically active metabolites which have been developed into drugs [8]. Vancomycin and rifampicin are antibiotics obtained from Actinomycetes strains against methicillin-resistant Staphylococcus aureus, tuberculosis, and leprosy [9]. Many antibiotics such as aminoglycosides, macrolides, β lactams, peptides, polyenes, tetracyclines, and anthracycline were obtained from the genus Streptomyces [10,11]. Whereas, gentamicin, loaiviticin A and B, and tetrocarcin antibiotics were isolated from Micromonospora strains [12,13].
Several antimicrobial compounds have been isolated from terrestrial or soil microorganisms. Between 2006 and 2007 alone, about 1,736 anti-infective, anticancer and antiinflammatory compounds like pestalone, hypoxysordarin, and equisetin were isolated from marine and aquatic environments [14,15]. However, the rate of finding new bioactive agents from this same environment for drug development has declined significantly [16]. e aquatic environment which comprises many living organisms including microbes is still considered an unexploited reservoir for novel bioactive compounds [17].
is has therefore necessitated the need to search for antimicrobial agents by focusing on antimicrobe-producing microbes from water bodies and soil samples. Hence, the aim of this study was to isolate, identify, and screen the metabolites of antimicrobe-producing microbes from selected soil and water bodies in Ghana.

Sampling.
irty-six (36) samples comprising sea water, river water, and soil samples were collected from three different sources in Ghana, namely, Dompoase landfill site, Kumasi, Ashanti region; Physics Garden, Kwame Nkrumah University of Science and Technology, Kumasi, Ashanti region; Kosiko River, Swedru, Central Region; and Ada Foah seashore, Ada, Greater Accra region. ese samples were transported on ice to the microbiological laboratory of the Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana, and stored in a refrigerator. e soil samples were collected at depths of 2-12 cm with a spatula into sterile plastic bags, while the river and seawater samples were collected into sterile 150 mL bottles [18].

Isolation of Pure Colonies from Samples.
(1.0 mL) of the water samples was separately pipetted into 9 mL sterile water making a ten-fold dilution. A 100 μL aliquot of samples was spread on 20 mL solidified nutrient agar plates. For soil samples, 1.0 g of the soil was suspended in 9 mL sterile water and diluted 5 times. One hundred microlitres (100 μL) of the suspension was drawn from all dilutions and spread onto separate 20 mL solidified nutrient agar plates (CM0003, Oxoid UK). All the plates were incubated at 37°C (using Incubator 3032-3033, Hamburg, Germany) for 72 hours. After three days of incubation, the culture plates were observed and microbial colonies surrounded with clear zones of inhibition were fished out. All isolates were separately cultured on a sterile nutrient agar slant and stored in a 30% sterile glycerol broth at −4°C in a frost-free refrigerator. e test organisms were prepared by streaking all test bacteria on 20 mL sterile nutrient agar and fungus strain on Sabouraud dextrose agar plates followed by incubation at 37°C for 24 h and 25°C for 72 h, respectively. Well-isolated colonies of each test organism were fished and suspended in 10 mL sterile water in test tubes. e turbidity of the suspensions was adjusted to that of the 0.5 McFarland standard.

Primary Screening of Isolated Microorganisms for Antimicrobial Activity.
e microbial isolates were evaluated by screening for the production of antimicrobial metabolites using the agar well diffusion method [19]. All the isolated organisms were cultured in 10 mL nutrient broths for eleven days at 37°C. e growth cultures were centrifuged for 15 minutes at 700 revolutions per minute and their supernatants were evaluated for antimicrobial activity against the test microorganisms whose inoculates were prepared and adjusted to the 0.5 McFarland standard. One hundred microliters (100 μL) of the test microbial suspension were inoculated into sterile nutrient agar plates by the pour plate method. Using a sterile cork-borer, five equidistant wells (10 mm in diameter) were created in each agar plate and filled with 200 μL of the microbial supernatant. e plates were kept for 2 h at room temperature for the diffusion of the bioactive metabolite into the plated agar. Two replicates were done and the plates were incubated for 24 h at 37°C. e diameter of zones of growth inhibition produced was measured and mean values were calculated. e isolate which showed the most promising activity (isolate DO5) was selected for further studies.

Biochemical and Morphological Characterization of
Isolate DO5 2.5.1. Macroscopic Characterization. e isolate was grown on nutrient agar plates at 30°C for 24 h and its phenotypic characteristics such as color and motility were determined [20]. DO5 colonies appeared gray.

Microscopic Characterization by Gram Staining.
Gram's staining was conducted to determine micromorphological characteristics of isolate DO5 using a DM700 light microscope fitted with a camera [21,22]. Isolate DO5 was grown on a plate of nutrient agar by streaking and incubating at 37°C for 24 h. A colony was picked to prepare a smear on a microscope slide (Sure friend Medicals Middlesex, England). e smear was sun-dried for 30 minutes and fixed to the slide by passing it through a Benson burner flame. It was stained with ammonium oxalate crystal violet solution for 60 seconds and rinsed with distilled water. e smear was flooded with Lugol's iodine solution for 30 seconds and washed with distilled water, and then decolorized with 70% alcohol and rinsed with distilled water. e smear was counterstained with a safranin solution for 1 minute, rinsed again with water, and blotted with a filter paper. e smear was observed under the microscope in oil immersion using × 100 objective lens [21].

Biochemical Reaction.
e pure isolate of DO5 was taken through a series of biochemical tests as indicated in Table 1.

Cultivation of Isolate DO5 on Selective Media.
Twenty-four-hour culture of isolate DO5 was streaked on sterile plates of cetrimide agar, bismuth sulfite Agar, Mac-Conkey agar, and mannitol salt agar. e plates were incubated for 24 h at 37°C for growth. where six pure colonies of the isolate were picked and suspended in 2 mL of 0.85% NaCl solution. e suspension was adjusted to the turbidity of the 0.5 McFarland standard prior to mixing with AUX-medium. e tube section from nitrate (NO 3 ) to p-nitrophenyl-β-D-galactopyranoside (PNPG) was filled with the saline solution and 200 μL of isolate DO5 culture. e tube and cupule from glucose (GLU) to phenyl-acetate (PAC) were also filled with AUXsuspension. Sterile mineral oil was added to glucose (GLU), arginine (ADH), and urea (URE) cupules after inoculation to create an anaerobic system and the strip was grown for 18 h at 28°C. e results were read and compared to the API standardized interpretation database.

MALDI-TOF Analysis.
e standard ethanol-formic acid (EFA) method was used to prepare isolate DO5 for the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). Following the protocol for standard ethanol-formic acid (EFA), isolate DO5 was grown on brain heart infusion agar for 24 h at 30°C. About fifteen (15) pure colonies were picked with sterile disposable plastic inoculating loop into 300 μL sterile distilled water in 2 mL Eppendorf tubes and vortexed. Nine hundred microliters (900 μL) of 96% ethanol were added and thoroughly mixed. e uniform mixture was centrifuged at a speed of 13,000 r.p.m. (revolutions per minute) for 2 minutes and the supernatant was separated from the microbial pellet by pipetting. e pellets were dried at 25°C for 2 minutes and suspended in a 150 μL mixture of 50% acetonitrile and 1% aqueous trifluoroacetic acid in a 2 mL Eppendorf tube containing 20 mg of acid-washed glass beads. e cells were vortexed and centrifuged again for 2 minutes to obtain the extract. One microliter (1 μL) of the extract was sported on MALDI-TOF steel target plate in replicates, dried at 25°C, and then covered with 1 μL of matrix solution (10 mg cyano-4-hydroxycinnamic acid in 1 mL sterile distilled water, 2.5% trifluoroacetic acid, 50% acetonitrile) for co-crystallization of the DNA. e DNA was analyzed with a MALDI-TOF MS spectrometer and the results were interpreted using Bruker Maldi Biotyper Flex control software [24,25].

Determination of the Effect of Incubation Period on Antimicrobial Activity of DO5 Metabolites.
e incubation period for highest metabolites production and maximum activity of DO5 was determined by fermenting the isolate in 360 mL of nutrient broth in a shaking incubator at 37°C for 18 days. After every 24 h intervals, 20 mL aliquots of the culture were pipetted into falcon tubes of 45 mL and centrifuged at 700 r.p.m. for 15 minutes. e cell-free supernatant culture was analyzed for antimicrobial activity against Klebsiella pneumoniae using the agar well diffusion method as described in the primary screening of isolated microorganisms for antimicrobial activity protocol above. e extract of isolate DO5 was assessed for antimicrobial activity against the test organisms. In the agar well diffusion method, two concentrations (50 mg/mL and 100 mg/mL) of the extract were prepared and tested against S. aureus, S. pyogenes, S. typhi, K. pneumoniae, P. mirabilis, V. cholera, E. faecalis, E. coli, and C. albicans in triplicate determination. Ciprofloxacin (12.5 μg/μL) and clotrimazole (25 μg/μL) were used as positive controls [19].

Broth Dilution
Method. DO5 extract was analyzed with a broth dilution method to determine the minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of the extract in 96 -well microtiter plates. Two-fold dilutions of 100 mg/mL to 0.78 mg/mL concentrations were prepared from the extract. One hundred microliters (100 μL) of the double strength nutrient broth and 80 μL of the extract were dispensed into 96-well plates followed by the addition of 20 μL of the test microbes' suspension (1.0 × 10 −5 CFU/ mL) to make a final volume of 200 μL. All the plates were incubated for 24 h at 37°C after which 20 μL of 1.25 mg/mL of tetrazolium salt (3-(4,5-dimethylthiazole-2yl-2,5-diphenyltetrazolium bromide) solution was added and incubated at 37°C for 30 min. Wells that developed a purple color after addition of the MTT indicated growth of test microbes and those that retained the yellow color indicated growth inhibition of the test microbes [19]. One hundred (100) microliters of cultures were pipetted from wells that showed inhibitory activity and subcultured into sterilized nutrient broths by incubation at 37°C for 24 h. e least concentration did not show microbial growth after the incubation period was considered the minimum bactericidal concentration.

Chromatographic Analyses of the Isolate DO5 Extract
2.11.1. in-Layer Chromatography. DO5 extract of 10 mg/ mL in methanol was profiled along the horizontal line drawn on a TLC plate of dimensions 2 × 8 cm.
e plates were developed in a petroleum ether-chloroform-methanol (80 : 10 : 10) solvent system and viewed under a UV lamp at a wavelength of 254 and 365 nm. e spots were circled with pencil and plates were sprayed with 1 mL anisaldehyde solution, air-dried, and heated at 100°C for 5 minutes in an oven for visibility of spots. Distance travelled by the spots and the solvent front were measured in cm with the aid of a meter ruler and used to calculate retardation factor (R f ) values.  4 Scientifica source were maintained at 250°C and 150°C, respectively. Detection of GC-MS was operated in electron impact mode with ionization energy of 70 electronvolts (eV) using helium gas of 99.99% purity as a carrier gas at a stable flow rate of 1 mL/min. e mass spectrum of the bioactive fractions was read at 70 eV, scanned within 0.5 seconds, and fragmented from 45 to 450 Da following their mass to charge ratio. e separated ions were detected by the Turbo-Mass detector and amplified signals of the spectra to Turbo-Mass version-6.1.0 software for recording the chromatogram. e solvent was started at zero minute and delayed for 2 minutes before the GC-MS was run for 47 minutes to initialize the system. e mass spectra of the bioactive fractions determined were interpreted and compared to the GC-MS spectrum database of the National Institute Standard and Technology (NIST).

Infrared Spectroscopic Analysis of DO5 Bioactive
Fraction. An attenuated total reflectance infrared spectrophotometer was used to determine the number of functional groups present in the DO5 bioactive fraction (BF2124). Ten milligrams (10 mg) of dried fractions were mounted directly on the KBr disc of the PerkinElmer 200 UATR (FT-IR C9413) spectrophotometer [29] and scanned through the IR region between the ranges of 400 to 4000 cm −1 at a resolution of 4 cm −1 .

Statistical Analysis. Data were analyzed with GraphPad
Prism version 8.0 for Windows (GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA followed by Dunnett's post hoc test was conducted.

Isolation and Antimicrobial Screening.
A total of 48 isolates suspected of having the capacity to produce antimicrobial metabolites were isolated from 36 samples collected, out of which only 10 showed zones of growth inhibition ranging from 12.50 ± 0.707 to 21.50 ± 0.701 mm against at least one of the test organisms used (  Table 2). In all, isolate DO5 exhibited a broad spectrum of activity against Gram-positive and Gram-negative bacteria including fungi and was selected for further studies.

Morphological and Biochemical Characterization of Isolate DO5.
Gram's staining and microscopic examination presented isolate DO5 as Gram-negative rod-like bacteria that reacted with 3% KOH solution to produce a stringy or mucoid reaction. e isolate grew in 1 to 3% NaCl solution but not in 5 to 7% w/v. It also showed growth from pH 7 to 9 but not at pH 4 to 6. e isolate grew on MacConkey agar, bismuth sulfite agar, cetrimide agar, and Sabouraud agar but not on mannitol salt agar. e isolate was a motile bacterium which did not produce indole, H 2 S gas, and VP but produced acid, oxidase, catalase, methyl red, and citrate (Table 3).

API 20 NE and MALDI-TOF Identification of Isolate DO5.
All values corresponding to the positive reactions in each group were added to generate a seven-digit number (7). Based on the numbered profile of API 20 NE analytical index book, isolate DO5 was identified to be P. aeruginosa (ID ≥ 99.9% and T ≥ 0.9). e isolate identification was confirmed to the species level by matrix-assisted laser.

Effect of Carbon Sources, Potassium Nitrate, Calcium Carbonate, and Glucose Concentrations on Antimicrobial Activity of Isolate DO5.
e isolate produced metabolites in maltose, galactose, sucrose, glucose, fructose, mannitol, lactose, and glycerol media which exhibited activity against S. aureus, E. coli, K. pneumoniae, and C. albicans. Metabolites in the xylose medium produced no activity against the organisms ( Figure S1). e bioactive metabolites produced in the potassium nitrate medium showed growth inhibitory action against the test organisms at all concentrations. e highest activity was observed at concentrations between 0.1 and 0.5% ( Figure S2). e metabolites produced in the CaCO 3 medium also exhibited activity against all test organisms at concentrations of 0.1-1.0% ( Figure S3). Bioactive metabolites produced by DO5 in the glucose medium showed activity against the test organisms at concentrations of 0.1-1.0% with Candida albicans showing the least activity ( Figure S4).

Incubation Period and Fermentation.
Isolate DO5 produced antimicrobial metabolites for maximum activity from day 3 with the highest zone of growth inhibition of 23.67 mm occurring on day 11. Whereas, the least activity of 14.33 mm was recorded on day 18 against K. pneumoniae ( Figure 1). Aliquots of isolate DO5 fermented supernatant which were taken every 24 h produced inhibitory activity of 17.00 ± 1.41, 17.59 ± 2.99, 17.50 ± 0.71 and 18.00 ± 00 mm against S. aureus, C. albicans, K. pneumoniae, and E. coli, respectively, in agar well determination (Table 4).

Extraction and Antimicrobial Activity of the Crude DO5
Extract.

Discussion
e growing negative impact of antimicrobial resistance has necessitated the search for newer and effective microbial agents from varying sources including microorganisms. River bodies and landfill sites have been reported to be major sites for obtaining antibiotic-producing bacteria. is study therefore reports the isolation of antibiotic-producing organisms from soil and water samples taken from the Dompoase landfill site, KNUST Physic Garden, Kosiko River, and Ada Foah seashore. Ten isolates were identified to be antibiotic-producing organisms. is could be due to the ecological role such as defensive mechanism to maintain their niche, or invasion of an established microbial community [30]. It has been reported that live and heat-killed cells of S. aureus induced the production of secondary metabolites from marine microbial isolates [31]. About 119 microorganisms were screened and only 27 isolates were found active against one test organism [18]. Fifty-seven (57) bacteria isolates were screened and only 2 showed activity [32]. In another investigation, 23 bacteria isolates tested for antimicrobial activity and only 7 isolates were active [33]. In this current study, ten (10) out of 48 isolates exhibited antimicrobial activity against the test microorganisms. e supernatant culture of isolate DO5 exhibited activity against the test organisms which indicated that metabolites are not produced only in the existence of competitions but can be produced in a medium that contains appropriate nutrients and at favorable temperature [32,34]. e activity of metabolites produced by isolate DO5 started on the 3 rd day of incubation and increased daily to the 11 th day. Many factors control the production of secondary metabolites and the principal factor among them is the formulation ingredients of the medium [32,34]. Differences that may occur in the fermentation media can result in changes in the size of inhibition zones produced by the metabolites. Isolate DO5 was found to produce high antimicrobial activity against test microbes in the fermentation media enriched with maltose,   8 Scientifica galactose, sucrose, glucose, fructose lactose, mannitol, and glycerol. From this study, isolate DO5 was morphologically reported as Gram-negative, rod-shaped, motile, and oxidasepositive and grew on all the selective media except mannitol salt agar because it does not tolerate high salt concentration. DO5 was indole-negative but catalase-positive. Pseudomonas aeruginosa, AZ-SH-B8, was identified using API 20 NE kit during the production of antibiotic sparsomycin and recorded similar results such as nitrate reduction activity, negative indole production, and urease and oxidase production [35]. Isolate DO5 was identified to the species level by MALDI-TOF mass spectrometry as Pseudomonas aeruginosa score value was greater than or equal to 2.35 [36,37]. e score value represents the identification of isolate DO5 to the species level. e extract of isolate DO5 had activity on all the test organisms. e MIC recorded ranged between 3.12 and 25 mg/mL whereas the MBC between 6.25 and 50 mg/mL. e TLC analysis revealed eight spots of bioactive compounds from the extract with different retardation factors. An infrared spectrum of the DO5 bioactive fraction showed some characteristic peaks due to the presence of functional groups such as C-O, C�O, C-H, and C�C. From the infrared studies on purification and physiochemical characterization of pyomelanin pigment produced from local Pseudomonas aeruginosa isolates showed presence of similar (C�O, C-H and C�C) functional groups [38]. e absorption peaks of the pigment resembled peaks of the bioactive DO5 fraction but pyomelanin was not found in the DO5 fraction which may be due to differences in geographical locations. Gas chromatography-mass spectrophotometry analysis conducted by Altaee et al. [39] identified oxime-, methoxy-phenyl, Edulan II, methyl-4-nitromethyl-4qpiperidinol, acetamide, N-methyl-N-4-2-fluoromethyl-1-pyrrolidyl-2-buty, oxaspiro 4,4 nonane-4-one, 2-isopropyl, octahydrochromen-2-one, 3,7-diazabicyclo 3.3.1 nonane, 9,9-dimethyl, N-3-N-aziridyl propylidene tetrahydrofurfurylamine, benzenemethanol-aminopropoxy-3-methyl, dithiocarbamate, dl-Allo-cystathionine, deoxyspergualin, and dl-2,6-diaminoheptanedioic acid from Pseudomonas aeruginosa isolated from urinary tract infection patients. None of these compounds were found in the DO5 bioactive fraction which may be due to  Antifungal, antimicrobial, and antioxidant [37,38] Scientifica 9 differences in their geographical locations. Results from the current GC-MS analysis of the DO5 fraction suggested bioactive compounds such as E-15-heptadecenal, 1-docosene, 3-eicosene, 1-eicosanol, 1-nonadecene, 1-hexadecanol, pyrrolizidine (pyrrolo) 1,2-a pyrazine-1,4-dione, 1-hexadecene, and 1-heptacosanol. e presence of these compounds could be responsible for the antimicrobial activity of DO5. Data Availability e datasets used and/or analyzed during the current study are included within the article. Further clarification can be obtained from the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest.